Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. While individual microRNAs (miRNAs) can control PH modestly, related miRNAs may conspire to regulate multiple target gene networks for a more robust influence on disease. Yet, the network biology of miRNAs has been poorly explored in vivo. We hypothesize that the miR-130/301 family is a pathogenic lynchpin controlling numerous target pathways (beyond pro-proliferative signaling) for broad control over PH. We will define mechanisms connecting the miR-130/301 family to pulmonary vascular dysfunction in vivo - via possible targeting of PPARg and TIMP2 to alter vasomotor tone and vascular stiffness. We will also delineate individual actions of miRNA family members and their coordinated effects in vivo. Thus, the redundant/ synergistic effects thought to be active in miRNA biology will be examined for the first time in PH in vivo. Specific Aims: Aim 1) Determine the importance of PPARg and endothelin-1 for control of pulmonary vasomotor tone by miR-130/301. To prove whether miR-130/301 causes vasoconstriction via regulating PPARg and endothelin-1 (ET-1), miR-130/301, PPARg, and ET-1 will be modulated in mice using systems of RNAi and adeno-associated viral (AAV) transgene delivery to the pulmonary vessels, along with intravital pulmonary vascular microscopy and pressure-flow curve analyses to measure vasomotor tone directly. Aim 2) Determine the importance of PPARg and TIMP2 for control of matrix deposition and pulmonary vascular stiffness by miR-130/301. We postulate that miR-130/301 further controls PH manifestation via repressing both PPARg and TIMP2, thus increasing matrix deposition and vascular stiffness. We will modulate miR-130/301, PPARg, and TIMP2 in mice, followed by biophysical (atomic force microscopy) and molecular assessment of pulmonary vascular stiffness. Results could provide the first evidence in vivo of miRNA-specific regulation of pulmonary vascular stiffness and combinatorial control of PH via multiple related miRNA targets. Aim 3) Define the coordinated actions of the miR-130/301 family members on overall PH manifestation. We postulate that the miR-130/301 family employs both synergism and redundancy to promote PH. An optimized system of miRNA delivery to the pulmonary vasculature in vivo will be used systematically to interrogate the individual versus integrated contributions of each miR-130/301 family member to PH manifestation in mice. This study will be the first to define the specific network-based parameters of miRNA biology in control of PH in vivo and could be a necessary guide in developing miRNA-based therapies of PH. Significance: Our proposal incorporates a rigorous expertise in miRNA biology with new technological advancements in the molecular, biophysical, and physiological study of PH in vivo. Thus, we aim to establish miR-130/301 as a multi-faceted regulator of PH -- offering new therapeutic targets and perhaps accelerating discovery in other diseases that rely upon complex miRNA networks for control of resultant pathophenotypes.